JP4146124B2 - Hepatitis virus propagation method and apparatus - Google Patents

Description

【００２５】
実施例２
実施例１と条件を変えて同様な実験を行った。
（１） 血清添加培養液での培養系 バイオリアクターは実施例１と同じものを用いた。フラスコにて培養した１×１０^９個のＦＬＣ４細胞を培地制御槽に播種した。２％血清添加培地（培養液）５０ｍｌ／日を用いて培養したところ、バイオリアクター内のＦＬＣ４細胞の酸素消費量は徐々に増加を始め、３０日目には２５ｐｐｍに（図３（Ａ）参照）、１０５日目には３５ｐｐｍに達した。経過中、バイオリアクター後方の溶存酸素濃度が１．０ｐｐｍ未満にならないように、培養温度を３７℃より徐々に低下させ（図３（Ａ）参照）、１０５日目には３０℃まで下げた。培養液中のアルブミン量は、低温培養でも経過中７５μｇ／ｍｌ以上であり、細胞の活動性は維持されていた。
培養における、温度（℃）、酸素濃度（ｐｐｍ）及びアルブミン濃度（μｇ／ｍｌ）の推移を図３（Ａ）に示す。図３（Ａ）の白丸印（○）は温度（℃）を示し、黒丸印（●）は酸素濃度（ｐｐｍ）を示し、白四角印（□）はアルブミン濃度（μｇ／ｍｌ）を示す。温度（℃）、及び酸素濃度（ｐｐｍ）は左側の目盛りで示され、アルブミン濃度（μｇ／ｍｌ）は右側の目盛りで示されている。横軸は日数を示している。数値は図３（Ｃ）に示されているとおりである。
前記した実施例１（５）と同様の方法により、排出培養液中のＨＣＶの検出を行った。
その結果を図３（Ｂ）に示す。図３（Ｂ）の白丸印（○）はＲＮＡ力価（ｔｉｔｅｒ）を示し、白四角印（□）はＨＣＶ−コア蛋白質が陰性であることを示し、黒四角印（■）はＨＣＶ−コア蛋白質が陽性であることを示す。図３（Ｂ）の縦軸はＲＮＡ力価（ｌｏｇ_１０コピー数／ｍｌ）であり、横軸は培養日数である。
図３（Ｃ）は、培養液中のＧＰＴ（ＩＵ／ｌ）、ＧＯＴ（ＩＵ／ｌ）、及びＬＤＨ（ＩＵ／ｌ）の推移を示す。図３（Ｃ）の黒丸印（●）はＧＰＴ（ＩＵ／ｌ）を示し、白丸印（○）はＧＯＴ（ＩＵ／ｌ））を示し、白四角印（□）はＬＤＨ（ＩＵ／ｌ）を示す。ＧＰＴ（ＩＵ／ｌ）、及びＧＯＴ（ＩＵ／ｌ）は左側の目盛りで示され、ＬＤＨ（ＩＵ／ｌ）は右側の目盛りで示されている。横軸は日数を示している。
【００２６】
（２） 無血清培養液での培養系 無血清培地（培養液）５０ｍｌ／日を用いてＦＬＣ４細胞をバイオリアクターで培養した。
結果を図４及び図５に示す。図５は、タイプ１ａの感染性クローンＲＮＡをバイオリアクターにトランスフェクションした場合（下記の（４）参照）のものである。
培養における、温度（℃）、酸素濃度（ｐｐｍ）及びアルブミン濃度（μｇ／ｍｌ）の推移を図４（Ａ）及び図５（Ａ）に示す。但し、図４（Ａ）にはアルブミン濃度（μｇ／ｍｌ）は示されていない。図４（Ａ）及び図５（Ａ）の白丸印（○）は温度（℃）を示し、黒丸印（●）は酸素濃度（ｐｐｍ）を示し、図５（Ａ）の白四角印（□）はアルブミン濃度（μｇ／ｍｌ）を示す。温度（℃）、及び酸素濃度（ｐｐｍ）は左側の目盛りで示され、アルブミン濃度（μｇ／ｍｌ）は右側の目盛りで示されている。横軸は日数を示している。数値は図４（Ｃ）又は図５（Ｃ）に示されているとおりである。
前記した実施例１（５）と同様の方法により、各排出培養液中のＨＣＶの検出を行った。
その結果を図４（Ｂ）及び図５（Ｂ）に示す。図４（Ｂ）及び図５（Ｂ）の白丸印（○）はＲＮＡ力価（ｔｉｔｅｒ）を示し、白四角印（□）はＨＣＶ−コア蛋白質が陰性であることを示し、黒四角印（■）はＨＣＶ−コア蛋白質が陽性であることを示す。図４（Ｂ）及び図５（Ｂ）の縦軸はＲＮＡ力価（ｌｏｇ_１０コピー数／ｍｌ）であり、横軸は培養日数である。
図４（Ｃ）及び図５（Ｃ）は、培養液中のＧＰＴ（ＩＵ／ｌ）、ＧＯＴ（ＩＵ／ｌ）、及びＬＤＨ（ＩＵ／ｌ）の推移を示す。図４（Ｃ）及び図５（Ｃ）の黒丸印（●）はＧＰＴ（ＩＵ／ｌ）を示し、白丸印（○）はＧＯＴ（ＩＵ／ｌ））を示し、白四角印（□）はＬＤＨ（ＩＵ／ｌ）を示す。ＧＰＴ（ＩＵ／ｌ）、及びＧＯＴ（ＩＵ／ｌ）は左側の目盛りで示され、ＬＤＨ（ＩＵ／ｌ）は右側の目盛りで示されている。横軸は日数を示している。
この培養系においては、培養温度は培養開始５日目に３５℃に下げて培養し、１００日以上の長期にわたり継代することなしに培養することができた（図５（Ａ）参照）。その間、細胞の酸素消費量は１５〜２０ｐｐｍで安定していた（図５（Ａ）参照）。
以上のように、ラジアルフロー型バイオリアクターを用いて３０〜３５℃の低温で培養することで、無血清培地または２％血清添加培地で、肝細胞癌由来細胞株を１００日以上の長期にわたり、継代することなしに培養することができ、その間、酸素消費量、グルコース消費量、アルブミン量等で示される肝細胞の活動性は保たれていた。
【００２７】
（３） 慢性肝炎患者血清による感染実験 実施例１の（４）と同様にして慢性肝炎患者血清を感染させ、実施例１（５）と同様にして排出培養液中のＨＣＶを検出した。その結果、感染後３日目にウイルスＲＮＡは一度陰性になったが、その後再び陽性化し、感染後１０日目に１０^３〜１０^４コピー／ｍｌと最大となった（図４（Ｂ）参照）。
このように、実施例１の場合と同様、接種後一度ＨＣＶＲＮＡは陰性になってから再び陽性化し増えてくることから、ウイルスが感染増殖しているものと考えられた。なお、実施例１及び２のいずれの感染実験でも、経過中ＧＯＴ、ＧＰＴ、ＬＤＨ等肝障害の指標の上昇は認められなかった（図４（Ｃ）及び図５（Ｃ）参照）。
【００２８】
（４） 感染性クローンによるトランスフェクション実験 タイプ１ａの感染性クローンＲＮＡをバイオリアクターにトランスフェクションしたところ（図５参照）、ウイルスＲＮＡはトランスフェクション直後から徐々に減少し、４４日目には１０^２コピー／ｍｌ未満にまでなったものの、その後５７日目に再び１０^４コピー／ｍｌまで増加し（図５（Ｂ）参照）、１００日目まで約１０^３〜１０^４コピー／ｍｌと持続していた。なお、トランスフェクションは、感染性クローンＨ７７（Ｙａｎａｇｉ ｅｔ ａｌ．，１９９８，Ｐｒｏｃ Ｎａｔｌ Ａｃａｄ Ｓｃｉ Ｕ Ｓ Ａ．Ａｕｇ ５；９４（１６）：８７３８−４３）の１０μｇを、リポフェクチン法により、上記した患者血清の場合と同様に、一時的に循環を止めてトランスフェクションした。なお、リポフェクチン法によるトランスフェクションは具体的には次のように行った。
前述のＨ７７よりインビトロでＲＮＡを合成した１０ｕｇをＯｐｔｉＭＥＭ１ｍｌにリポフェクチン１５０ｕｌとともに混合し１５分間室温に放置した。その後、リアクター内をＯｐｔｉＭＥＭで十分満たした後、ＲＮＡを注入した。
また、培養上清中のコア蛋白も徐々に増加し、４４日目に最大になった（図５（Ｂ）参照）。コア蛋白は、免疫測定により定量した。より具体的に記載すると、沈殿試薬により培養液中のウイルス粒子を沈殿分画として収集後、それを分散試薬に分散させる。抗体試薬、および中和試薬で処理した後、ＨＣＶコア蛋白質がチューブ上の抗ＨＣＶコア蛋白質モノクローナル抗体に結合して、複合体を形成する。未反応物質を洗浄除去した後、ペルオキシダーゼ標識抗ＨＣＶコア蛋白質モノクローナル抗体を加えると、前述のチューブ上の複合体に結合する。未反応物質を洗浄除去した後、ＨＰＰＡ気質液を添加するとチューブ上に結合したペルオキシダーゼ酵素により蛍光物質が生成され、これを３２３ｎｍの励起光を照射し、生じた蛍光を４１０ｎｍで測定する。これをあらかじめ標準液より作製された検量線から濃度を算定する さらに、コア蛋白質の存在を確認するために、トランスフェクション後９６日目の培養液を２００倍に濃縮し、１０〜６０％（Ｗ／Ｗ）蔗糖勾配法にて分画して、それぞれの分画のＨＣＶ ＲＮＡとＨＣＶのコア蛋白質を測定した。
結果を図６に示す。図６の下段のグラフの白丸印（○）はＨＣＶ ＲＮＡの力価（ｌｏｇ_１０コピー数／ｍｌ）を示し、黒丸印（●）はＨＣＶコア蛋白質の濃度（ｐｇ／ｍｌ）を示し、図６の上段のグラフの黒四角印（■）はコア蛋白質の密度（ｇ／ｍｌ）を示す。
この結果、密度勾配が約１．０７及び１．１８ｇ／ｍｌの２箇所でコア蛋白質の極大が測定され、コア蛋白質が２峰性の曲線であることがわかった。このことは、培養液中にウイルス粒子が存在していることを示すものである。
また、トランスフェクション後８、４４日目の培養液をＲＮａｓｅ処理した後にｎｅｓｔ−ＲＴ ＰＣＲでＨＣＶのＲＮＡが検出できたことから、ウイルス粒子内に保護されたＨＣＶ ＲＮＡの存在が示唆された（図７参照）。なお、このｎｅｓｔ−ＲＴ ＰＣＲで用いたプライマーの塩基配列は、フォワード側がＣＴＧＴＧＡＧＧＡＡＣＴＡＣＴＧＴＣＴＴ，ＴＴＣＡＣＧＣＡＧＡＡＡＧＣＧＴＣＴＡＧ、リバース側が，ＡＡＣＡＣＴＡＣＴＣＧＧＣＴＡＧＣＡＧＴ、ＧＴＧＡＴＣＣＡＡＧＡＡＡＧＧＡＣＣＣであった。
結果を図７として示す。図７は左側から、培養前日（−１日）の培養液、培養８日目の培養液、培養４４日目の培養液、対照としての血清、対照としてのＲＮＡであり、各々、ＲＮａｓｅ処理無し（ＲＮａｓｅ−）でｎｅｓｔ−ＲＴ ＰＣＲ無し（ＲＴ−）、ＲＮａｓｅ処理無し（ＲＮａｓｅ−）でｎｅｓｔ−ＲＴ ＰＣＲ有り（ＲＴ＋）、及びＲＮａｓｅ処理有り（ＲＮａｓｅ＋）でｎｅｓｔ−ＲＴ ＰＣＲ有り（ＲＴ＋）の３つのレーンで構成されている。図７の上段の数字は培養日数を示し、＋−の表示の上の段はｎｅｓｔ−ＲＴ ＰＣＲ処理の有無を有り（＋）、無し（−）で示し、下の段はＲＮａｓｅ処理の有無を有り（＋）、無し（−）で示している。図７の縦方向の数値は塩基数（ｍｅｒ）を示す。
さらに、トランスフェクション後１１０日目の細胞内にタグを用いたＲＴ ＰＣＲ法でマイナス鎖ＲＮＡを検出できたことから、細胞内にウイルス複製中間体の存在が示唆された（図８参照）。このＲＴ ＰＣＲは、具体的には次のように行った。このＲＴ ＰＣＲで用いたプライマーの塩基配列は、逆転写反応に、ＴＣＴＴＧＧＴＧＧＣＧＡＡＴＡＡＧＣＣＡＴＧＧＣＧＴＴＡＧＴＡＴ，ＰＣＲ反応にフォワード側が、ＴＣＡＴＧＧＴＧＧＣＧＡＡＴＡＡ，リバース側が、ＣＧＣＧＧＣＡＡＣＡＡＧＴＡＡＡであった。
結果を図８に示す。図８のＭはマーカーを示し、レーン１のＮは細胞（−）で、ネガティブストランドＲＮＡ（ｎｅｇａｔｉｖｅ ｓｔｒａｎｄ ＲＮＡ）及びポジティブストランドＲＮＡ（ｐｏｓｉｔｉｖｅ ｓｔｒａｎｄ ＲＮＡ）も存在していないコントロールを示し、レーン２の（−）ＲＮＡはネガティブストランドＲＮＡ（ｎｅｇａｔｉｖｅ ｓｔｒａｎｄ ＲＮＡ）を加えた場合を、レーン３の（＋）ＲＮＡはポジティブストランドＲＮＡ（ｐｏｓｉｔｉｖｅ ｓｔｒａｎｄ ＲＮＡ）を加えた場合をそれぞれ示し、レーン４の細胞（Ｃｅｌｌ）はＲＦＢで培養したＨＣＶ感染細胞の場合を示す。図８の縦方向の数値は分子量を示す。この結果、培養された感染細胞中にネガティブストランドＲＮＡ（ｎｅｇａｔｉｖｅ ｓｔｒａｎｄ ＲＮＡ）の存在が認められた。
培養経過中のＨＶＲの塩基配列をもとの感染性クローンと比較したところ、２５、７１、１０６日目にそれぞれ１、２、２個の塩基の変異が認められたが、この培養経過を通して特定の塩基配列への収束、変異の蓄積などは認められなかった。結果を次の表２に示す。結果は、表２中の「ＲＦＢ（Ｒａｄｉａｌ Ｆｌｏｗ Ｂｉｏｒｅａｃｔｏｒ）」の欄に示されている。なお、配列はアミノ酸の１文字表記の配列で示されている。
表２中の「クローン数」は、培養液中から回収されたクローン数を示す。
表２中の「Ｄ」は感染後の日数を示す。
表２中のアミノ酸配列の上の数字は、ＨＣＶのタンパク質におけるアミノ酸の位置を示し、ハイフンは最上段に記載されているアミノ酸と同じであることを示す。
【００２９】
【表２】

【００３０】
以上のように、タイプ１ａの感染性クローンをバイオリアクターにトランスフェクションしたところ、１）ウイルスＲＮＡの再増加、２）コア蛋白質の増加、３）粒子内ウイルスＲＮＡの存在、４）細胞内ウイルス複製中間体の存在、５）塩基配列の変異、の５通りの方法でＨＣＶの複製を確認することに成功した。ウイルス増殖に伴う肝障害については、経過中明らかなＧＯＴ、ＧＰＴ、ＬＤＨの上昇などは認められなかった（図５（Ｃ）参照）。
【００３１】
【発明の効果】
本発明は、生体外では培養が困難であるとされていた肝炎ウイルスの生体外での培養、増殖方法を初めて提供するものであり、肝炎ウイルスの研究のみならず、肝炎ウイルス感染症の治療、予防、及び機構を研究開発するための材料を提供するものである。より具体的には、ウイルス性肝炎の治療薬の研究開発に必須とされているウイルスを簡便な方法で提供することができる手段を提供するものである。
さらに、本発明の方法は、ウイルスの増殖の機構及び変異の機構を解明するためのウイルスの増殖方法を提供するものである。
このように本発明は、ウイルスの生態の研究のみならず、ウイルス感染症の治療、予防、処置の方法を研究開発ためのウイルスの必要な量を安定に供給する方法を提供するものである。
【００３２】
【配列表】
SEQUENCE LISTING
<110> Seishi NAGAMORI
<120> Method for Proliferating Hepatitis Cirus and Apparatus Therefor
<130> JA904421
<150> JP 11-233647
<151> 1999-08-20
<160> 5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<223> primer used for synthesizing cDNA of hepatitic C virus by reverse transcription
<400> 1
aacactactc ggctagcagt 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<223> primer used for amplifying by PCR hepatitic C virus cDNA
<400> 2
ctgtgaggaa ctactgtctt 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<223> primer used for amplifying by PCR hepatitic C virus cDNA
<400> 3
aacactactc ggctagcagt 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<223> primer used for amplifying by PCR hepatitic C virus cDNA
<400> 4
ttcacgcaga aagcgtctag 20
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence
<223> primer used for amplifying by PCR hepatitic C virus cDNA
<400> 5
gtgatccaag aaaggaccc 19
【図面の簡単な説明】
【図１】 図１は、本発明の方法に用いられる培養装置の例としてのラジアルフロー型バイオリアクターの縦断面及び横断面を示す図である。
【図２】 図２は、本発明の培養システムの例を模式的に示す図である。
【図３】 図３は、血清添加培養液を用いた培養系における、温度（℃）、酸素濃度（ｐｐｍ）及びアルブミン濃度（μｇ／ｍｌ）の推移（図３（Ａ））、排出培養液中のＨＣＶの検出の結果（図３（Ｂ））、及び培養液中のＧＰＴ（ＩＵ／ｌ）、ＧＯＴ（ＩＵ／ｌ）、及びＬＤＨ（ＩＵ／ｌ）の推移（図３（Ｃ））を示したものである。
図３（Ａ）の白丸印（○）は温度（℃）を示し、黒丸印（●）は酸素濃度（ｐｐｍ）を示し、白四角印（□）はアルブミン濃度（μｇ／ｍｌ）を示す。温度（℃）、及び酸素濃度（ｐｐｍ）は左側の目盛りで示され、アルブミン濃度（μｇ／ｍｌ）は右側の目盛りで示されている。横軸は日数を示している。
図３（Ｂ）の白丸印（○）はＲＮＡ力価（ｌｏｇ_１０コピー数／ｍｌ）を示し、白四角印（□）はＨＣＶ−コア蛋白質が陰性であることを示し、黒四角印（■）はＨＣＶ−コア蛋白質が陽性であることを示し、横軸は培養日数である。
図３（Ｃ）の黒丸印（●）はＧＰＴ（ＩＵ／ｌ）を示し、白丸印（○）はＧＯＴ（ＩＵ／ｌ））を示し、白四角印（□）はＬＤＨ（ＩＵ／ｌ）を示す。ＧＰＴ（ＩＵ／ｌ）、及びＧＯＴ（ＩＵ／ｌ）は左側の目盛りで示され、ＬＤＨ（ＩＵ／ｌ）は右側の目盛りで示されている。横軸は日数を示している。
【図４】 図４は、無血清培養液を用いた培養系における、温度（℃）、及び酸素濃度（ｐｐｍ）の推移（図４（Ａ））、排出培養液中のＨＣＶの検出の結果（図４（Ｂ））、及び培養液中のＧＰＴ（ＩＵ／ｌ）、ＧＯＴ（ＩＵ／ｌ）、及びＬＤＨ（ＩＵ／ｌ）の推移（図４（Ｃ））を示したものである。
図４（Ａ）の白丸印（○）は温度（℃）を示し、黒丸印（●）は酸素濃度（ｐｐｍ）を示す。横軸は日数を示している。
図４（Ｂ）の白丸印（○）はＲＮＡ力価（ｌｏｇ_１０コピー数／ｍｌ）を示し、白四角印（□）はＨＣＶ−コア蛋白質が陰性であることを示し、黒四角印（■）はＨＣＶ−コア蛋白質が陽性であることを示し、横軸は培養日数である。
図４（Ｃ）の黒丸印（●）はＧＰＴ（ＩＵ／ｌ）を示し、白丸印（○）はＧＯＴ（ＩＵ／ｌ））を示し、白四角印（□）はＬＤＨ（ＩＵ／ｌ）を示す。ＧＰＴ（ＩＵ／ｌ）、及びＧＯＴ（ＩＵ／ｌ）は左側の目盛りで示され、ＬＤＨ（ＩＵ／ｌ）は右側の目盛りで示されている。横軸は日数を示している。
【図５】 図５は、無血清培養液を用いた培養系で、培養時にタイプ１ａの感染性クローンＲＮＡをバイオリアクターにトランスフェクションした場合における、温度（℃）、酸素濃度（ｐｐｍ）及びアルブミン濃度（μｇ／ｍｌ）の推移（図５（Ａ））、排出培養液中のＨＣＶの検出の結果（図５（Ｂ））、及び培養液中のＧＰＴ（ＩＵ／ｌ）、ＧＯＴ（ＩＵ／ｌ）、及びＬＤＨ（ＩＵ／ｌ）の推移（図５（Ｃ））を示したものである。
図５（Ａ）の白丸印（○）は温度（℃）を示し、黒丸印（●）は酸素濃度（ｐｐｍ）を示し、白四角印（□）はアルブミン濃度（μｇ／ｍｌ）を示す。温度（℃）、及び酸素濃度（ｐｐｍ）は左側の目盛りで示され、アルブミン濃度（μｇ／ｍｌ）は右側の目盛りで示されている。横軸は日数を示している。
図５（Ｂ）の白丸印（○）はＲＮＡ力価（ｌｏｇ_１０コピー数／ｍｌ）を示し、白四角印（□）はＨＣＶ−コア蛋白質が陰性であることを示し、黒四角印（■）はＨＣＶ−コア蛋白質が陽性であることを示し、横軸は培養日数である。
図５（Ｃ）の黒丸印（●）はＧＰＴ（ＩＵ／ｌ）を示し、白丸印（○）はＧＯＴ（ＩＵ／ｌ））を示し、白四角印（□）はＬＤＨ（ＩＵ／ｌ）を示す。ＧＰＴ（ＩＵ／ｌ）、及びＧＯＴ（ＩＵ／ｌ）は左側の目盛りで示され、ＬＤＨ（ＩＵ／ｌ）は右側の目盛りで示されている。横軸は日数を示している。
【図６】 図６は、トランスフェクション後９６日目の培養液を蔗糖勾配法にて分画して、それぞれの分画のＨＣＶ ＲＮＡとＨＣＶのコア蛋白質を測定した結果を示す。図６の下段のグラフの白丸印（○）はＨＣＶ ＲＮＡの力価（ｌｏｇ_１０コピー数／ｍｌ）を示し、黒丸印（●）はＨＣＶコア蛋白質の濃度（ｐｇ／ｍｌ）を示し、図６の上段のグラフの黒四角印（■）はコア蛋白質の密度（ｇ／ｍｌ）を示す。
【図７】 図７は、感染性クローンＨ７７をトランスフェクション後８、４４日目の培養液を、ＲＮａｓｅでの処理、及びｎｅｓｔ−ＲＴ ＰＣＲ法で検出した結果を示す図である。図７は左側から、培養前日（−１日）の培養液、培養８日目の培養液、培養４４日目の培養液、対照としての血清、対照としてのＲＮＡであり、各々、ＲＮａｓｅ処理無し（ＲＮａｓｅ−）でｎｅｓｔ−ＲＴ ＰＣＲ無し（ＲＴ−）、ＲＮａｓｅ処理無し（ＲＮａｓｅ−）でｎｅｓｔ−ＲＴ ＰＣＲ有り（ＲＴ＋）、及びＲＮａｓｅ処理有り（ＲＮａｓｅ＋）でｎｅｓｔ−ＲＴ ＰＣＲ有り（ＲＴ＋）の３つのレーンで構成されている。図７の上段の数字は培養日数を示し、＋−の表示の上の段はｎｅｓｔ−ＲＴ ＰＣＲ処理の有無を有り（＋）、無し（−）で示し、下の段はＲＮａｓｅ処理の有無を有り（＋）、無し（−）で示している。図７の縦方向の数値は塩基数（ｍｅｒ）を示す。
【図８】 図８は、感染性クローンＨ７７をトランスフェクションした後１１０日目の細胞内にタグを用いたストランド特異的ＲＴ−ＰＣＲ（Ｓｔｒａｎｄ−ｓｐｅｃｉｆｉｃ ＲＴ−ＰＣＲ）法でマイナス鎖ＲＮＡを検出した結果を示す図である。図８のＭはマーカーを示し、レーン１のＮは細胞（−）で、ネガティブストランドＲＮＡ（ｎｅｇａｔｉｖｅ ｓｔｒａｎｄ ＲＮＡ）及びポジティブストランドＲＮＡ（ｐｏｓｉｔｉｖｅ ｓｔｒａｎｄ ＲＮＡ）も存在していないコントロールを示し、レーン２の（−）ＲＮＡはネガティブストランドＲＮＡ（ｎｅｇａｔｉｖｅ ｓｔｒａｎｄ ＲＮＡ）を加えた場合を、レーン３の（＋）ＲＮＡはポジティブストランドＲＮＡ（ｐｏｓｉｔｉｖｅ ｓｔｒａｎｄ ＲＮＡ）を加えた場合をそれぞれ示し、レーン４の細胞（Ｃｅｌｌ）はＲＦＢで培養したＨＣＶ感染細胞の場合を示す。図８の縦方向の数値は分子量を示す。[0001]
BACKGROUND OF THE INVENTION
  The present invention relates to hepatitis C virus (HCV)ofHow to proliferateTo the lawConcerning.
[0002]
[Prior art]
  The cDNA of HCV was cloned in 1989, and then the structure and processing mechanism have been elucidated using various expression systems. As a result, a very effective diagnostic system was developed, and post-transfusion hepatitis due to HCV in Japan has been largely suppressed at present.
  As described above, the whole picture of HCV is becoming clear, but research at the gene level has preceded, and basic research such as elucidation of biology of virus replication, particle formation, mutation, and carcinogenic mechanisms is still progressing. However, the development of therapeutic methods using drugs such as HCV vaccines, protease inhibitors, and antisenses has not progressed. This is due to the fact that an in vitro HCV proliferation system does not yet exist. It is very difficult to grow HCV in cultured hepatocytes, and there has never been a report that this has been successful. Therefore, at present, there is only a method for using the chimpanzee in the above research. However, this is very expensive, and there are problems with individual differences and reproducibility. In addition, its use is limited from the point of animal welfare. From such a background, establishment of a proliferation system for hepatitis viruses such as HCV using cultured cells independent of clinical trials and animal experiments is desired.
[0003]
[Problems to be solved by the invention]
  Therefore, the object of the present invention is to cultureHumanHC using hepatocytesVProviding a method of propagation.
[0004]
[Means for Solving the Problems]
  As a result of earnest research, the present inventorHumanThe culture solution is circulated through the incubator containing the carrier carrying the hepatocytes.RuIn a dial flow bioreactorHumanCulturing hepatocytes, infecting them with HCV,HumanBy continuing the culture of hepatocytes, HCVThe present invention was completed by finding that it can be grown.
  That is, the present inventionThe human maintained in a radial flow type hepatocyte bioreactor in which a culture solution is circulated from a peripheral part of the bioreactor main body containing a human porous cell supported on a particulate porous carrier toward a central part. Hepatocytes are infected with hepatitis C virus, and then the human hepatocytes are cultured by circulating a culture solution from the peripheral part of the bioreactor body toward the center part, whereby the infected hepatitis C virus is A method for propagating hepatitis C virus, comprising growing in human hepatocytes, wherein the hepatitis C virus infection is performed by adding hepatitis C virus to the culture medium, After adding the hepatitis virus to the culture medium, circulate the used medium without supplying fresh medium, then stop the circulation of the culture liquid, and then without supplying fresh medium, By circulating medium use further comprising the step of culturing, the method of the proliferation of hepatitis C virusAbout. The present invention also provides:The human maintained in a radial flow type hepatocyte bioreactor in which a culture solution is circulated from a peripheral part of the bioreactor main body containing a human porous cell supported on a particulate porous carrier toward a central part. Hepatocytes are infected with hepatitis C virus, and then the human hepatocytes are cultured by circulating a culture solution from the peripheral part of the bioreactor body toward the center part, whereby the infected hepatitis C virus is A method for propagating hepatitis C virus, comprising growing in human hepatocytes, wherein the hepatitis C virus infection is performed by adding hepatitis C virus to the culture medium, A method for proliferating hepatitis C virus, wherein the supply rate of fresh medium and the oxygen supply rate are made larger than the previous rate before hepatitis virus is added to the culture medium.About.
[0006]
  BookAccording to the method of the invention, the HC grown in the discharged culture brothVObtainable. Thus, the present invention uses cultured cells and ex vivo HCVThis is the first time to provide a method for efficient growth.
  Therefore, the present invention not only predicts the effects of interferon that has been used in conventional treatments, but also HC.V waHC such as development of cutin, preparation of anti-HCV antibody, protease inhibitor, polymerase inhibitor and antisense drugVThis greatly contributes to the development of therapeutic drugs.
[0008]
  The present inventionUsed inIncubatorIs, Radial flow bioreactorIs. An example of a preferred radial flow bioreactor of the present invention will be described with reference to the drawings.
  FIG. 1 schematically shows an example of a preferred radial flow type bioreactor of the present invention. The upper diagram in FIG. 1 is a longitudinal sectional view of the radial flow bioreactor, and the lower diagram is a transverse sectional view of the radial flow bioreactor. The radial flow bioreactor 10 includes a cylindrical bioreactor body (incubator) 12. The outer peripheral wall of the bioreactor body 12 is made of a porous material having a large number of through holes, and the culture solution can flow from the outside to the inside of the bioreactor body through these through holes. The diameter of the through hole is smaller than the carrier particles described later, and is a size that can sufficiently supply the culture solution into the bioreactor body 12, and is usually preferably about 20 to 80 μm. A cylindrical casing 14 is provided so as to surround the outer side of the bioreactor body 12 so that the cross-section is concentric with the bioreactor body 12, and an outer peripheral wall of the bioreactor body 12 and the casing 14 An annular culture solution supply path 16 is formed between them. The culture solution supply path 16 communicates with the culture solution supply pipe 18 at the bottom thereof. At the center of the bioreactor body 12, a culture solution discharge pipe 20 is provided. The outer peripheral wall of the culture solution discharge pipe 20 is also made of a porous material having a large number of through-holes having the same size as that of the bioreactor body 12, and the culture solution can flow but the carrier cannot pass through.
[0009]
  Furthermore, a large number of particulate porous carriers 22 are accommodated inside the bioreactor body 12. The material of the porous carrier is not particularly limited, but a preferable example is a spherical porous glass bead. The diameter of the porous carrier is not particularly limited, but is preferably about 0.1 mm to 6 mm, and particularly preferably about 0.3 mm to 1.2 mm. The pore diameter in the carrier is not particularly limited, but is preferably about 10 to 300 μm, and particularly preferably about 20 to 120 μm. The porosity in the carrier particles is not particularly limited, but is preferably about 30 to 70%, and particularly preferably about 40 to 60%. Such a porous glass bead is commercially available from Schott Glasswerk Co. Ltd. under the trade name of Silane, and this commercially available product can be preferably used. Further, the density of the carrier particles accommodated in the bioreactor body 12 is not particularly limited, but it is preferable to pour the particles into the bioreactor body as much as possible under gravity.
  The size of the radial flow bioreactor is not particularly limited, and the volume in the bioreactor body 12 is usually about 5 ml to several tens of liters, but may be outside this range.
[0010]
  Next, a method for culturing hepatocytes using the above radial flow bioreactor will be described. The flow of the culture solution is indicated by arrows in FIG. That is, the culture solution is supplied from the bottom to the culture solution supply path 16 in the outer peripheral portion of the bioreactor body 12 through the culture solution supply pipe 18. The culture fluid flows upward through the culture fluid supply path 16, and enters the bioreactor main body 12 through a large number of through holes provided in the outer wall of the bioreactor main body 12. Then, it flows in the bioreactor main body 12 toward the center, enters the culture medium discharge pipe 20 from a large number of through holes provided in the culture liquid discharge pipe 20, and moves upward in the culture liquid discharge pipe 20. Then, it is discharged from the top of the culture medium discharge pipe 20 to the outside of the bioreactor body 12. In the radial flow type bioreactor shown in FIG. 1, the culture solution is supplied from the bottom, but the culture solution only has to flow from the outer periphery of the bioreactor body 12 toward the center. It is good also as a structure supplied from the top part of the liquid supply path 16. FIG. Further, it is preferable that a part of the discharged culture solution is circulated again and supplied as a culture solution. That is, as the culture solution, it is preferable to use a mixture of a fresh culture solution and a recycled culture solution. These mixing ratios are preferably automatically controlled so that the daily glucose consumption (g / day) / oxygen consumption (g / day) ratio is 0.5 to 15, particularly about 3 to 10. .
[0011]
  The culture solution used here may be of any composition as long as hepatocytes can be cultured and proliferated, and minerals, sugars, amino acids, peptides, vitamins, organic acids, nucleic acids, pH adjustment What is necessary is just to contain a component required for culture | cultivation of cells, such as an agent and oxygen. For example, inorganic materials include NaCl, KCl, MgCl₂, MgSO₄, NaH₂PO₄, FeSO₄-7H₂O, ZnSO₄-7H₂O, CuSO₄-5H₂Examples of such amino acids and peptides include L-asparagine hydrochloride, L-alanine, alanyl-L-glutamine, L-aspartic acid, L-glutamate, glycine, glycyl-L-glutamine, L-isoleucine, L -Lysine, L-phenylalanine, L-serine, L-ornithine, L-threonine, L-tryptophan, L-tyrosine, L-valine, insulin, etc. Examples of sugars include sugars, sugar alcohols, glycosides, etc. Examples thereof include D-glucose, D-mannose, D-galactose, and inositol. Examples of the organic acid include organic acid derivatives such as free acid or ester, and examples thereof include succinic acid, choline bitartrate, folic acid, sodium pyruvate, and glycerophosphoric acid. Vitamins include pyridoxal hydrochloride, riboflavin and the like. Examples of the nucleic acid include uridine. pH adjusters include NaOH, carbon dioxide, NaHCO₃Etc.
[0012]
  A commercially available culture solution can also be used as the culture solution. Moreover, what added about 1-3% of serum, such as fetal calf serum, to this can be used preferably. It is preferable to adjust the oxygen concentration and pH in the culture solution. It is preferable to adjust the oxygen concentration in the culture solution so that the oxygen concentration in the discharged culture solution is 1 ppm or more. That is, the oxygen supply amount is preferably 0.025 to 0.75 ml / min, particularly about 0.05 to 0.5 ml / min per 1 ml of the volume of the bioreactor body (incubator). The supply rate of the fresh culture solution is not particularly limited, but is usually about 0.25 to 100 ml / day, preferably about 0.5 to 50 ml / day per 1 ml of the volume of the bioreactor body (incubator). is there. The circulation rate of the culture solution is not particularly limited, but is usually about 0.25 to 2.0 L / day, preferably about 0.5 to 1.0 L / day per 1 ml of the volume of the bioreactor body (incubator). It is. The pH of the culture solution is preferably adjusted to about 7.0 using sodium hydroxide solution or carbon dioxide. Furthermore, the temperature of the culture solution is preferably adjusted to about 37 ° C. or lower.
[0013]
  Hepatocytes grow on the surface of the porous carrier and the inner surface of the pores in the porous carrier. The proliferated hepatocytes are carried on the surface of the porous carrier and the inner surface of the pores, and further filled in the voids between the porous carriers. Adhesion and proliferation of hepatocytes to a porous carrier can be achieved by supplying a hepatocyte suspension in the culture medium to the radial flow bioreactor as the culture medium. When the hepatocyte suspension is supplied as a culture solution, hepatocytes adhere to the surface of the porous carrier or the inner surface of the pores while the culture fluid flows while contacting the porous carrier, and proliferate there.
  The density of hepatocytes added to the culture medium at the start of culture is not particularly limited, but is usually 10⁵-10⁹About cells / ml, preferably 10⁶-10⁷The total number of hepatocytes added to the culture solution is appropriately selected according to the volume of the bioreactor body. For example, when the volume in the bioreactor body 12 is 200 ml, the total number of hepatocytes is usually 10⁷-10¹⁰About one, preferably 10⁸-10⁹About one is appropriate. In addition, after the culture solution to which hepatocytes have been added has spread throughout the bioreactor body, the flow of the culture solution is stopped for about 3 hours to 12 hours to prevent the hepatocytes from flowing out of the bioreactor body and to support hepatocytes. It is preferred to promote adhesion to the top.
[0014]
  The hepatocytes used areHuman hepatocytes,A sample collected from a human liver by necropsy or the like may be grown by culturing using a known plate culture method or the like, but in order to reliably achieve growth and maintenance in a bioreactor over a long period of time.HumanIt is preferable to use an established cell line of hepatocytes.HumanEstablished hepatocyte cell lines are known per se, and any cell line can be used. Examples of preferred cell lines include FLC-4 (U.S. Pat. No. 5,804,441, deposited with FERM BP-5165 at the Biotechnology Institute of Technology), HepG2 (available from ATCC), Huh7 (Japanese). (Available from Cancer Research Resources Bank (JCRB)), FLC-1, FLC-2, FLC-3, FLC-5, FLC-6 and FLC-7 (the cell lines of these FLC series are described in K. Fujise, S .; Nagamori, H. Kameda et al., HEPATOLOGY, 8: 1425, 1988; 158-162, 1998; Kaw da, M. et al., In Vitro Cell. Dev. Biol., 34: 109-115, 1998: and Tetsu Hasumura et al. See Artificial Blood, 5, 33-37, 1997, etc.) However, it is not limited to these. When the present inventor compared the proliferative ability of HCV with respect to a plurality of types of hepatic cell lines, HCV proliferated best in FLC-4 cells. Therefore, it is preferable to use FLC-4 cells. In FLC-4 cells, it is considered that there are some host factors that specifically stabilize HCV minigene RNA and increase translation efficiency.
[0015]
  If the cells are usually cultured for 5 to 15 days after the supply of the hepatocytes, the hepatocytes sufficiently proliferate and become a preferable state for infecting the hepatitis virus. Hepatocytes are about 10 in the bioreactor body.⁸Grows to more than cells / ml. When the volume in the bioreactor body 12 is 200 ml, the total number of hepatocytes is about 2.9 × 10.¹⁰To grow.
  After proliferating hepatocytes,C typeInfect with hepatitis virus. BookAccording to the method of the invention, DifferentIt is also possible to simultaneously propagate a plurality of viruses of the same strain type.C typeInfection with hepatitis virus can be performed, for example, by supplying serum from a chronic hepatitis patient in the supply culture medium and supplying the culture medium.C typeThe possibility of infection can be further increased by directly adding a culture solution containing hepatitis virus to the hepatocytes in the bioreactor body. The amount of hepatitis patient serum to be supplied is not particularly limited, but about 1/50 to 1/10 of the volume of the bioreactor body is appropriate. OrC typeHepatitis virus can be constructed in hepatocytes,C typeHepatitis virus infectious cDNA clones can also be injected. Therefore, in the present invention, “C typeInfecting the hepatitis virus is a completeC typeNot only infecting hepatitis virus particles, but also in hepatocytesC typeInfecting an infectious recombinant vector that expresses a nucleic acid capable of constructing a hepatitis virus is also included. In addition,C typeAfter supplying the hepatitis virus-containing culture solution, the supply of a new culture solution and the circulation of the culture solution are preferably stopped for about 2 to 24 hours, more preferably about 2 to 10 hours, and then preferably for 2 hours. The culture medium discharged from the top of the bioreactor body 12 is supplied again to the bioreactor body 12 as a culture liquid without supplying a new culture liquid for about 48 hours, more preferably about 6 to 48 hours. preferable. By doing thisC typeThe possibility of infection with hepatitis virus can be increased. Also,C typeImmediately before infection with hepatitis virus, about 15 minutes to 4 hours, more preferably about 30 minutes to 2 hours, the fresh medium supply rate and oxygen supply rate up to that time are about 1.5 to 4 times, more preferably 1.5 It is preferable to increase the culture to about 2.5 to 2.5 times. By doing thisC typeThe possibility that hepatitis virus is infected can be increased and the state of the cells can be kept good. In addition, after the start of culturing hepatocytes, when the oxygen consumption is about ppm ± 30% of the volume of the bioreactor body (for example, 15 ppm ± 30% when the volume in the bioreactor body is 30 ml) In order to increase the probability of virus infection, it is preferable to infect the virus as described above after gradually lowering the culture temperature and stabilizing the oxygen consumption. At this time, the culture temperature is preferably 28 ° C to 34 ° C, more preferably about 29 ° C to 32 ° C. In addition, it is preferable to carry out the culture after virus infection at such a low temperature in order to sustain the virus infection and keep the cell state in good condition.
[0016]
  aboveC typeAfter hepatitis virus infection treatment, by continuing the culture of hepatocytes under the conditions described above,C typeIn the culture medium in which hepatitis virus grows in the hepatocytes and is discharged from the culture medium discharge tube 20 about 2-3 weeks after infection.C typeHepatitis virus is included. Therefore, from the discharged culture solutionC typeBy collecting hepatitis virusC typeHepatitis virus can be isolated. From the cultureC typeSeparation of hepatitis virus can be performed by conventional methods such as filtration using an ultrafiltration membrane, centrifugation, gel filtration chromatography, etc..
  BookThe virus propagated by the method of the invention can be used as an immunogen for developing vaccines and inducing anti-hepatitis virus antibodies. In the above cultured hepatocytesC typeHepatitis virus propagation systemC typeIn addition to recovering hepatitis virus, it can also be used to develop hepatitis therapeutic agents such as protease inhibitors, antisense RNA, and antisense DNA.
[0017]
【Example】
  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
[0018]
Example 1
(1) Radial flow type bioreactor A radial flow type bioreactor having the structure shown in FIG. 1 was prepared. The bioreactor body 12 and the culture solution discharge pipe 20 were made of a porous sintered metal material having a through hole having a diameter of about 40 μm. The volume in the bioreactor body 12 was 30 ml. The porous carrier filled in the bioreactor body 12 was porous glass beads (trade name Siran, Schott Glasswerk Co. Ltd., Germany). The diameter of this glass bead is 0.6mm, the inside is a hole like a honeycomb, the porosity is 50%, the surface area is 90m²/ L-matrix, pore diameter was 20 to 120 μm. 18 g of such glass beads were filled in the bioreactor body 12. Therefore, the cell adhesion area is 1500cm.²/ G-matrix.
[0019]
(2) Culture System An outline of a culture system including the radial flow bioreactor of (1) is schematically shown in FIG. In the system shown in FIG. 2, a fresh culture solution is stored in a fresh culture solution storage container 24 and is transferred into a culture solution adjustment tank 28 by a pump 26. The culture solution adjusting tank 28 is provided with a stirrer 30 where the oxygen concentration, carbon dioxide concentration and pH of the culture solution are adjusted. In addition, the heating means is provided in the stand 31 which mounts the culture solution adjustment tank 28, and the temperature of a culture solution can be adjusted. A 1N NaOH solution is stored in the NaOH storage container 32, and is transferred to the culture solution adjustment tank 28 by a pump 34 as necessary, thereby adjusting the pH of the culture solution. On the other hand, an oxygen cylinder 36 and a carbon dioxide cylinder 38 are connected to the culture solution adjustment tank 28 via a flow rate controller 40. A necessary amount of oxygen and carbon dioxide is supplied from the oxygen cylinder 36 and the carbon dioxide cylinder 38 to the culture solution adjustment tank 28, respectively. The flow rate controller 40 is connected to a microcomputer 42, and the flow rate is checked and controlled at a rate of once every 20 minutes by the microcomputer. The culture solution whose oxygen concentration, carbon dioxide concentration and pH are adjusted in the culture solution adjusting tank 28 is supplied from the bottom to the radial flow bioreactor 10 by the pump 44. The supplied culture solution is discharged from the top of the culture solution discharge pipe of the radial flow type bioreactor 10 as described with reference to FIG. The discharged culture solution is stored in the discharge culture solution storage container 48 by the pump 46. In the culture system shown in FIG. 2, a path for returning the discharged culture solution to the medium adjustment tank 28 is also provided, and all or part of the discharged culture solution is supplied again as the culture solution as necessary. Is also possible. Although not shown, the microcomputer 42 is connected to each pump and measures an oxygen concentration meter (not shown) that measures a culture solution supplied to the bioreactor body and an oxygen concentration of the discharged culture solution. Connected to an oxygen concentration meter (not shown) and further connected to a pH meter and a thermometer (not shown) provided in the culture solution adjusting tank 28. The oxygen concentration, pH, temperature, and supply amount of the culture solution are as follows. It is automatically controlled by the microcomputer 42.
[0020]
(3) Seeding and culture of hepatocytes 2 × 10 5 of the FLC-4 strain, which is an established hepatocyte cell line⁹Subculture was performed in a flask up to a single cell. After circulating the culture solution in the bioreactor body, FLC-4 cells cultured in the flask were added to the culture solution and supplied into the bioreactor body to inoculate hepatocytes. Six hours after seeding, the pumps 44 and 46 were stopped to prevent the cells from flowing out of the bioreactor body. Thereafter, a fresh culture solution was supplied at a flow rate of 25 ml / day. Moreover, the circulation rate of the culture solution was 10 to 40 L / day. The pH of the culture solution was 7.0, the temperature was 37 ° C., and the oxygen concentration was automatically controlled by a computer so that the oxygen concentration in the drained culture solution was 1 to 6 ppm.
The culture solution has the following composition (all units are mg / L).
NaCl 6000, KCl 400,
MgCl₂                100, MgSO₄                  98,
NaH₂PO₄            125, FeSO₄-7H₂O 0.8,
ZnSO₄-7H₂O 0.01, CuSO₄-5H₂O 0.001,
D-glucose 2000, D-mannose 500,
L-asparagine hydrochloride 200, D-galactose 200,
L-alanine 20, alanyl-L-glutamine 500,
L-aspartic acid 20, L-glutamate 20,
Glycine 30, glycyl-L-glutamine 500,
L-isoleucine 105, L-lysine 146,
L-phenylalanine 67, L-serine 80,
L-ornithine 100, L-threonine 95,
L-tryptophan 25, L-tyrosine 64,
L-valine 94, succinic acid 106,
Uridine 5, Choline ditartaric acid 20,
Folic acid 4, inositol 20,
Pyridoxal hydrochloride 4, riboflavin 0.4,
Sodium pyruvate 110, glycerophosphoric acid 1500,
HEPES 1200, NaHCO₃            1800,
Human transferrin 5, insulin 5,
Phenol red 5.
What added 2% of fetal bovine serum to this was used. The activity of the cells was confirmed by the increase of oxygen and glucose consumption in the culture medium, and the increase of oxygen consumption was observed smoothly. Here, the oxygen consumption was determined from the difference between the oxygen concentration in the culture solution supplied to the bioreactor body 12 and the oxygen concentration in the culture solution discharged from the top of the bioreactor body 12. The glucose consumption was determined by measuring the glucose concentration in the discharged culture solution using a commercially available glucose concentration measurement kit and calculating the difference between this concentration and the glucose concentration in the supplied culture solution.
[0021]
(4) Infection with HCV The oxygen consumption reached 15 ppm on the 7th day from the start of the culture in the reactor, and at this point, the culture temperature was gradually lowered to 32 ° C. Infection with HCV was performed on the 9th day from the start of the culture when the oxygen consumption of cells was stable. Prior to HCV infection, the culture fluid supply and oxygen supply were doubled and cultured for 1 hour. Thereafter, infection with HCV was performed. Infection with HCV is carried out from a culture solution circulation tube immediately after the top of the bioreactor body using 1 ml of serum of a chronic hepatitis C patient (infection titer against chimpanzee is 5.5 CID50 / ml) dissolved in 10 ml of culture solution. This was performed by branching and supplying the bioreactor body from a pipe (not shown) communicating with the bioreactor body. Thereafter, the pump was stopped for 6 hours. Next, the circulation pump 44 was started, but the entire amount of the culture solution discharged from the top of the bioreactor 10 was returned to the culture solution preparation tank 28, and cultured for 24 hours without supplying a new medium. Thereafter, the culture was continued as described above (however, the culture temperature was 29 to 32 ° C.).
[0022]
(5) Detection of HCV in discharged culture solution After HCV infection treatment, the culture was resumed under the normal conditions described above, and HCV in the discharged culture solution was detected by RT-PCR, which is a routine method, every day.
Here, the base sequence of the primer used for reverse transcription is
  AACACTACTCGGCTAGCAGT,
In addition, the base sequence of the primer used for PCR is the first
  CTGTGAGGAACTACTGTCTT, and
  AACACTACTCGGCTAGCAGT, the second time,
  TTCACGCAGAAAGCGTCTAG, and
  It was GTGATCCAAGAAAGGACCC.
  PCR was carried out for 35 cycles with a total volume of 50 μl, a denaturation step at 94 ° C. for 45 seconds, an annealing step at 55 ° C. for 45 seconds, and an extension step at 70 ° C. for 60 seconds.
As a result, HCV was detected 1 to 2 days after the infection treatment, but it was not detected after that, and it was detected again from the 16th day.⁴-10⁵The maximum was copy / ml.
  HCV RNA was detected all the way up to 100 days after infection. The reason that HCV was detected 1 to 2 days after the infection treatment is considered to be that from which the added HCV had flowed out. It is considered that the outflow of the added HCV was terminated because it was not detected from the third day after the infection treatment. And it is considered that HCV infected with hepatocytes proliferated in the hepatocytes and the proliferated HCV was released into the culture medium because it was detected again after the 16th day.
[0023]
(6) Base sequence of HCV When the base sequence of HVR (hypervariable region, hyper variable region) of the virus detected on the 23rd day after infection was examined (see the following Table 1), it was mostly 95% in the bioreactor. Were originally identical to the major clone Al, 55-60% in patient serum.
  The results are shown in Table 1 so that they can be compared with blood transfusion examples and chimpanzee examples. “RFB (Radial Flow Bioreactor)” in Table 1 indicates the result of the experiment. The sequence is shown as a one-letter amino acid sequence.
  “Number of clones” in Table 1 indicates the number of clones recovered from sera of donors, chimpanzees, and recipients, and recovered from RAD. The number of clones recovered from blood donor and chimpanzee sera is based on data from Aizaki et al.
  In Table 1, “W” indicates the number of weeks after blood transfusion, and “D” indicates the number of days after infection. The number above the amino acid sequence in Table 1 indicates the position of the amino acid in the HCV protein, and the hyphen is the same as the amino acid described at the top.
[0024]
[Table 1]

[0025]
Example 2
A similar experiment was performed under the same conditions as in Example 1.
(1) Culture system with serum-added culture solution The same bioreactor as in Example 1 was used. 1 × 10 cultured in flask⁹FLC4 cells were seeded in a medium control tank. When cultured using 50 ml / day of a 2% serum-supplemented medium (culture solution), the oxygen consumption of FLC4 cells in the bioreactor started to increase gradually and reached 25 ppm on the 30th day (see FIG. 3 (A)). ) And reached 105 ppm on the 105th day. During the course, the culture temperature was gradually decreased from 37 ° C. (see FIG. 3 (A)) so that the dissolved oxygen concentration behind the bioreactor did not become less than 1.0 ppm, and lowered to 30 ° C. on the 105th day. The amount of albumin in the culture broth was 75 μg / ml or more during the course of low temperature culture, and the cell activity was maintained.
  The changes in temperature (° C.), oxygen concentration (ppm) and albumin concentration (μg / ml) in the culture are shown in FIG. In FIG. 3A, white circles (◯) indicate temperature (° C.), black circles (●) indicate oxygen concentration (ppm), and white squares (□) indicate albumin concentration (μg / ml). Temperature (° C.) and oxygen concentration (ppm) are shown on the left scale, and albumin concentration (μg / ml) is shown on the right scale. The horizontal axis indicates the number of days. The numerical values are as shown in FIG.
  HCV in the effluent culture was detected by the same method as in Example 1 (5) described above.
  The result is shown in FIG. In FIG. 3 (B), a white circle mark (◯) indicates RNA titer (titer), a white square mark (□) indicates that HCV-core protein is negative, and a black square mark (■) indicates HCV-core. Indicates that the protein is positive. In FIG. 3 (B), the vertical axis represents RNA titer (log₁₀Copy number / ml), and the horizontal axis represents the number of days of culture.
  FIG. 3C shows changes in GPT (IU / l), GOT (IU / l), and LDH (IU / l) in the culture solution. In FIG. 3C, the black circle (●) indicates GPT (IU / l), the white circle (◯) indicates GOT (IU / l), and the white square (□) indicates LDH (IU / l). Indicates. GPT (IU / l) and GOT (IU / l) are shown on the left scale, and LDH (IU / l) is shown on the right scale. The horizontal axis indicates the number of days.
[0026]
(2) Culture system in serum-free culture solution FLC4 cells were cultured in a bioreactor using a serum-free medium (culture solution) 50 ml / day.
  The results are shown in FIGS. FIG. 5 shows the case where type 1a infectious clone RNA was transfected into a bioreactor (see (4) below).
  Changes in temperature (° C.), oxygen concentration (ppm) and albumin concentration (μg / ml) in the culture are shown in FIGS. 4 (A) and 5 (A). However, the albumin concentration (μg / ml) is not shown in FIG. In FIG. 4A and FIG. 5A, the white circle mark (◯) indicates the temperature (° C.), the black circle mark (●) indicates the oxygen concentration (ppm), and the white square mark (□ in FIG. 5A). ) Indicates albumin concentration (μg / ml). Temperature (° C.) and oxygen concentration (ppm) are shown on the left scale, and albumin concentration (μg / ml) is shown on the right scale. The horizontal axis indicates the number of days. The numerical values are as shown in FIG. 4 (C) or FIG. 5 (C).
  HCV in each effluent culture was detected by the same method as in Example 1 (5) described above.
  The results are shown in FIGS. 4B and 5B. In FIG. 4 (B) and FIG. 5 (B), the white circle mark (◯) indicates the RNA titer (titer), the white square mark (□) indicates that the HCV-core protein is negative, and the black square mark ( (2) indicates that HCV-core protein is positive. The vertical axis of FIG. 4 (B) and FIG. 5 (B) is the RNA titer (log).₁₀Copy number / ml), and the horizontal axis represents the number of days of culture.
  4 (C) and 5 (C) show changes in GPT (IU / l), GOT (IU / l), and LDH (IU / l) in the culture solution. In FIG. 4C and FIG. 5C, the black circle mark (●) indicates GPT (IU / l), the white circle mark (◯) indicates GOT (IU / l)), and the white square mark (□) indicates LDH (IU / l) is shown. GPT (IU / l) and GOT (IU / l) are shown on the left scale, and LDH (IU / l) is shown on the right scale. The horizontal axis indicates the number of days.
  In this culture system, the culture temperature was lowered to 35 ° C. on the 5th day from the start of the culture, and the culture could be performed without subculture for a long period of 100 days or longer (see FIG. 5 (A)). Meanwhile, the oxygen consumption of the cells was stable at 15 to 20 ppm (see FIG. 5A).
  As described above, by culturing at a low temperature of 30 to 35 ° C. using a radial flow type bioreactor, a hepatocellular carcinoma-derived cell line can be obtained over a long period of 100 days or more in a serum-free medium or a medium supplemented with 2% serum. The cells could be cultured without being subcultured, and the activity of the hepatocytes indicated by oxygen consumption, glucose consumption, albumin amount, etc. was maintained during that time.
[0027]
(3) Infection experiment with chronic hepatitis patient serum Infecting chronic hepatitis patient serum in the same manner as in Example 1 (4), and detecting HCV in the effluent culture solution in the same manner as in Example 1 (5). As a result, the viral RNA became negative once on the 3rd day after infection, but then became positive again, and 10 days on the 10th day after infection.³-10⁴The copy / ml was the maximum (see FIG. 4B).
  Thus, as in Example 1, since HCV RNA became negative once after inoculation and then became positive again and increased, it was considered that the virus was infecting and proliferating. In any of the infection experiments of Examples 1 and 2, there was no increase in indicators of liver damage such as GOT, GPT, and LDH during the course (see FIGS. 4C and 5C).
[0028]
(4) Transfection experiment using infectious clones When infectious clone RNA of type 1a was transfected into a bioreactor (see Fig. 5), viral RNA gradually decreased immediately after transfection, and 10 days on the 44th day.²10 copies again on the 57th day after reaching less than copy / ml⁴Increased to copy / ml (see FIG. 5 (B)), about 10 until day 100³-10⁴Persisted at copy / ml. Transfection was performed using 10 μg of the infectious clone H77 (Yanagi et al., 1998, Proc Natl Acad Sci USA Aug 5; 94 (16): 8738-43) by the lipofectin method described above. As in, the circulation was temporarily stopped and transfection was performed. Specifically, transfection by the lipofectin method was performed as follows.
  10 ug of RNA synthesized in vitro from the aforementioned H77 was mixed with 1 ml of OptiMEM together with 150 ul of lipofectin and left at room temperature for 15 minutes. Thereafter, after the reactor was sufficiently filled with OptiMEM, RNA was injected.
  In addition, the core protein in the culture supernatant gradually increased and reached the maximum on day 44 (see FIG. 5B). Core protein was quantified by immunoassay. More specifically, the virus particles in the culture solution are collected as a precipitate fraction by the precipitation reagent, and then dispersed in the dispersion reagent. After treatment with the antibody reagent and neutralizing reagent, the HCV core protein binds to the anti-HCV core protein monoclonal antibody on the tube to form a complex. After washing away unreacted substances, a peroxidase-labeled anti-HCV core protein monoclonal antibody is added to bind to the complex on the aforementioned tube. After washing away unreacted substances and adding HPPA gas, a fluorescent substance is generated by the peroxidase enzyme bound on the tube, and this is irradiated with 323 nm excitation light, and the resulting fluorescence is measured at 410 nm. The concentration is calculated from a calibration curve prepared in advance from a standard solution. Further, in order to confirm the presence of the core protein, the culture solution at 96 days after transfection is concentrated 200 times, and 10-60% (W / W) Fractionation was performed by a sucrose gradient method, and HCV RNA and HCV core protein of each fraction were measured.
  The results are shown in FIG. The white circle (◯) in the lower graph of FIG. 6 indicates the titer (log) of HCV RNA.₁₀Copy number / ml), black circles (●) indicate HCV core protein concentration (pg / ml), black squares (■) in the upper graph of FIG. 6 indicate core protein density (g / ml) Indicates.
  As a result, the maximum of the core protein was measured at two locations where the density gradient was about 1.07 and 1.18 g / ml, and it was found that the core protein was a bimodal curve. This indicates that virus particles are present in the culture solution.
  In addition, HCV RNA could be detected by nesto-RT PCR after RNase treatment of the culture solution on day 8 and 44 after transfection, suggesting the presence of protected HCV RNA in the virus particles (FIG. 7). In addition, the base sequence of the primer used in this next-RT PCR was CGTTGAGGAACTACTGTTCTT, TTCACGCAGAAAAGCGTCTAG on the forward side, and AACACTACTCGGCTAGCAGT, GTGATCCCAAGAAAGGACCC on the reverse side.
  The results are shown as FIG. FIG. 7 shows, from the left side, the culture solution the day before culture (-1 day), the culture solution on the 8th day of culture, the culture solution on the 44th day of culture, serum as a control, and RNA as a control, each without RNase treatment. (RNase−) without next-RT PCR (RT−), without RNase treatment (RNase−), with next-RT PCR (RT +), and with RNase treatment (RNase +), with next-RT PCR (RT +) 3 It consists of two lanes. The numbers in the upper part of FIG. 7 indicate the number of days of culture, the upper part of the display of + − indicates the presence / absence of the next-RT PCR treatment (+), and the absence (−), and the lower part indicates the presence / absence of the RNase treatment. With (+) and without (-). The numerical value in the vertical direction in FIG. 7 indicates the number of bases (mer).
  In addition, minus-strand RNA could be detected by RT PCR using a tag in the cells 110 days after transfection, suggesting the presence of viral replication intermediates in the cells (see FIG. 8). Specifically, this RT PCR was performed as follows. The base sequences of the primers used in this RT PCR were TCTTGGTGGGCGAATAAGCCATGGCGTAGTAGAT for the reverse transcription reaction, TCATGGTGGCGAATAAA for the PCR reaction, and CGCGGCCAACAAGTAAA for the reverse side.
  The results are shown in FIG. In FIG. 8, M represents a marker, N in lane 1 represents a cell (−), a control in which neither negative strand RNA (negative strand RNA) nor positive strand RNA (positive strand RNA) was present, and lane 2 ( -) RNA represents the case where negative strand RNA (negative strand RNA) was added, (+) RNA in lane 3 represents the case where positive strand RNA (positive strand RNA) was added, and the cell (Cell) in lane 4 represents The case of HCV-infected cells cultured with RFB is shown. The numerical value in the vertical direction in FIG. 8 indicates the molecular weight. As a result, the presence of negative strand RNA was observed in the cultured infected cells.
  Comparison of the base sequence of HVR during the course of culture with the original infectious clone revealed mutations of 1, 2, and 2 bases on days 25, 71, and 106, respectively. Convergence to the nucleotide sequence and accumulation of mutations were not observed. The results are shown in Table 2 below. The results are shown in the column of “RFB (Radial Flow Bioreactor)” in Table 2. The sequence is shown as a one-letter amino acid sequence.
  “Number of clones” in Table 2 indicates the number of clones recovered from the culture medium.
  “D” in Table 2 indicates the number of days after infection.
  The number above the amino acid sequence in Table 2 indicates the position of the amino acid in the HCV protein, and the hyphen is the same as the amino acid described at the top.
[0029]
[Table 2]

[0030]
  As described above, when a type 1a infectious clone was transfected into a bioreactor, 1) viral RNA increased again, 2) core protein increased, 3) presence of intraparticle viral RNA, and 4) intracellular viral replication. We succeeded in confirming the replication of HCV by the five methods of existence of intermediate and 5) mutation of base sequence. Regarding liver damage associated with virus growth, no apparent increase in GOT, GPT, LDH, etc. was observed during the course (see FIG. 5C).
[0031]
【The invention's effect】
  The present invention provides for the first time a method for in vitro culture and propagation of hepatitis virus, which has been difficult to culture in vitro, not only for hepatitis virus research, but also for treatment of hepatitis virus infection, It provides materials for research and development of prevention and mechanism. More specifically, the present invention provides a means by which a virus that is essential for research and development of a therapeutic drug for viral hepatitis can be provided by a simple method.
  Furthermore, the method of the present invention provides a virus propagation method for elucidating the mechanism of virus propagation and mutation.
  As described above, the present invention provides a method for stably supplying a necessary amount of virus for research and development of a method for treatment, prevention and treatment of viral infections as well as research on the ecology of viruses.
[0032]
[Sequence Listing]
                          SEQUENCE LISTING
<110> Seishi NAGAMORI
<120> Method for Proliferating Hepatitis Cirus and Apparatus Therefor
<130> JA904421
<150> JP 11-233647
<151> 1999-08-20
<160> 5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<223> primer used for synthesizing cDNA of hepatitic C virus by reverse transcription
<400> 1
aacactactc ggctagcagt 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<223> primer used for amplifying by PCR hepatitic C virus cDNA
<400> 2
ctgtgaggaa ctactgtctt 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<223> primer used for amplifying by PCR hepatitic C virus cDNA
<400> 3
aacactactc ggctagcagt 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<223> primer used for amplifying by PCR hepatitic C virus cDNA
<400> 4
ttcacgcaga aagcgtctag 20
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence
<223> primer used for amplifying by PCR hepatitic C virus cDNA
<400> 5
gtgatccaag aaaggaccc 19
[Brief description of the drawings]
FIG. 1 is a view showing a longitudinal section and a transverse section of a radial flow bioreactor as an example of a culture apparatus used in the method of the present invention.
FIG. 2 is a diagram schematically showing an example of the culture system of the present invention.
FIG. 3 shows changes in temperature (° C.), oxygen concentration (ppm) and albumin concentration (μg / ml) in a culture system using a serum-added culture solution (FIG. 3 (A)), excretion culture solution. Of detection of HCV in blood (FIG. 3 (B)), and changes in GPT (IU / l), GOT (IU / l), and LDH (IU / l) in the culture medium (FIG. 3 (C)) Is shown.
  In FIG. 3A, white circles (◯) indicate temperature (° C.), black circles (●) indicate oxygen concentration (ppm), and white squares (□) indicate albumin concentration (μg / ml). Temperature (° C.) and oxygen concentration (ppm) are shown on the left scale, and albumin concentration (μg / ml) is shown on the right scale. The horizontal axis indicates the number of days.
  The white circle (◯) in FIG. 3 (B) indicates the RNA titer (log₁₀Copy number / ml), white square (□) indicates that HCV-core protein is negative, black square (■) indicates that HCV-core protein is positive, and the horizontal axis indicates culture. Is the number of days.
  In FIG. 3C, the black circle (●) indicates GPT (IU / l), the white circle (◯) indicates GOT (IU / l), and the white square (□) indicates LDH (IU / l). Indicates. GPT (IU / l) and GOT (IU / l) are shown on the left scale, and LDH (IU / l) is shown on the right scale. The horizontal axis indicates the number of days.
FIG. 4 shows changes in temperature (° C.) and oxygen concentration (ppm) (FIG. 4 (A)) and results of detection of HCV in the effluent culture medium in a culture system using a serum-free culture medium. (FIG. 4 (B)) and changes in GPT (IU / l), GOT (IU / l), and LDH (IU / l) in the culture medium (FIG. 4 (C)).
  In FIG. 4A, white circles (◯) indicate temperature (° C.), and black circles (●) indicate oxygen concentration (ppm). The horizontal axis indicates the number of days.
  The white circles (Ｂ) in FIG. 4 (B) indicate the RNA titer (log₁₀Copy number / ml), white square (□) indicates that HCV-core protein is negative, black square (■) indicates that HCV-core protein is positive, and the horizontal axis indicates culture. Is the number of days.
  In FIG. 4C, the black circle (●) indicates GPT (IU / l), the white circle (◯) indicates GOT (IU / l), and the white square (□) indicates LDH (IU / l). Indicates. GPT (IU / l) and GOT (IU / l) are shown on the left scale, and LDH (IU / l) is shown on the right scale. The horizontal axis indicates the number of days.
FIG. 5 shows temperature (° C.), oxygen concentration (ppm), and albumin when a type 1a infectious clone RNA was transfected into a bioreactor in a culture system using a serum-free culture medium. Changes in concentration (μg / ml) (FIG. 5A), results of detection of HCV in the drained culture solution (FIG. 5B), GPT (IU / l), GOT (IU / I) in the culture solution 1) and the transition of LDH (IU / l) (FIG. 5C).
  In FIG. 5A, white circles (◯) indicate temperature (° C.), black circles (●) indicate oxygen concentration (ppm), and white squares (□) indicate albumin concentration (μg / ml). Temperature (° C.) and oxygen concentration (ppm) are shown on the left scale, and albumin concentration (μg / ml) is shown on the right scale. The horizontal axis indicates the number of days.
  The white circles (O) in FIG. 5B indicate the RNA titer (log).₁₀Copy number / ml), white square (□) indicates that HCV-core protein is negative, black square (■) indicates that HCV-core protein is positive, and the horizontal axis indicates culture. Is the number of days.
  In FIG. 5C, black circles (●) indicate GPT (IU / l), white circles (◯) indicate GOT (IU / l), and white squares (□) indicate LDH (IU / l). Indicates. GPT (IU / l) and GOT (IU / l) are shown on the left scale, and LDH (IU / l) is shown on the right scale. The horizontal axis indicates the number of days.
FIG. 6 shows the results obtained by fractionating the culture solution 96 days after transfection by the sucrose gradient method and measuring the HCV RNA and HCV core protein in each fraction. The white circle (◯) in the lower graph of FIG. 6 indicates the titer (log) of HCV RNA.₁₀Copy number / ml), black circles (●) indicate HCV core protein concentration (pg / ml), black squares (■) in the upper graph of FIG. 6 indicate core protein density (g / ml) Indicates.
FIG. 7 is a diagram showing the results of detection of the culture solution 8 and 44 days after transfection of infectious clone H77 by treatment with RNase and the next-RT PCR method. FIG. 7 shows, from the left side, the culture solution the day before culture (-1 day), the culture solution on the 8th day of culture, the culture solution on the 44th day of culture, serum as a control, and RNA as a control, each without RNase treatment. (RNase−) without next-RT PCR (RT−), without RNase treatment (RNase−), with next-RT PCR (RT +), and with RNase treatment (RNase +), with next-RT PCR (RT +) 3 It consists of two lanes. The numbers in the upper part of FIG. 7 indicate the number of days of culture, the upper part of the display of + − indicates the presence / absence of the next-RT PCR treatment (+), and the absence (−), and the lower part indicates the presence / absence of the RNase treatment. With (+) and without (-). The numerical value in the vertical direction in FIG. 7 indicates the number of bases (mer).
FIG. 8 shows detection of minus-strand RNA by strand-specific RT-PCR (Strand-specific RT-PCR) method using a tag in cells 110 days after transfection of infectious clone H77. It is a figure which shows a result. In FIG. 8, M represents a marker, N in lane 1 represents a cell (−), a control in which neither negative strand RNA (negative strand RNA) nor positive strand RNA (positive strand RNA) was present, and lane 2 ( -) RNA represents the case where negative strand RNA (negative strand RNA) was added, (+) RNA in lane 3 represents the case where positive strand RNA (positive strand RNA) was added, and the cell (Cell) in lane 4 represents The case of HCV-infected cells cultured with RFB is shown. The numerical value in the vertical direction in FIG. 8 indicates the molecular weight.

Claims (5)

The human maintained in a radial flow type hepatocyte bioreactor in which a culture solution is circulated from a peripheral part of the bioreactor main body containing a human porous cell supported on a particulate porous carrier toward a central part. Hepatocytes are infected with hepatitis C virus, and then the human hepatocytes are cultured by circulating a culture solution from the peripheral part of the bioreactor body toward the center part, whereby the infected hepatitis C virus is A method for propagating hepatitis C virus, comprising growing in human hepatocytes, wherein the hepatitis C virus infection is performed by adding hepatitis C virus to the culture medium, After adding the hepatitis virus to the culture medium, circulate the used medium without supplying fresh medium, then stop the circulation of the culture liquid, and then without supplying fresh medium, Further comprising, a method for proliferating hepatitis C virus the step of culturing by circulating medium use. The human maintained in a radial flow type hepatocyte bioreactor in which a culture solution is circulated from a peripheral part of the bioreactor main body containing a human porous cell supported on a particulate porous carrier toward a central part. Hepatocytes are infected with hepatitis C virus, and then the human hepatocytes are cultured by circulating a culture solution from the peripheral part of the bioreactor body toward the center part, whereby the infected hepatitis C virus is A method for propagating hepatitis C virus, comprising growing in human hepatocytes, wherein the hepatitis C virus infection is performed by adding hepatitis C virus to the culture medium, A method for propagating hepatitis C virus, wherein the supply rate of fresh medium and the oxygen supply rate are made larger than the previous rate before the hepatitis virus is added to the culture solution. After the hepatitis C virus was added to the culture medium, the used medium was circulated without supplying a fresh medium, then the culture medium was stopped, and then used without supplying a fresh medium. The method according to claim 2, further comprising the step of culturing by circulating the medium. The method according to any one of claims 1 to 3, wherein the human hepatocytes are an established cell line. The method according to claim 4, wherein the established cell line is a FLC-4 strain (FERM BP-5165).

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